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Aldolase mutant

A ‐phosphate aldolase and transformant technology, applied in the field of bioengineering, can solve the problems of substrate concentration limitation, product separation difficulty, DERA enzyme inactivation, etc., and achieve the effect of reducing the addition amount

Active Publication Date: 2013-11-27
ABIOCHEM BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0005] Wong [3,4] et al. obtained the chiral side chain of statins by one-pot continuous aldol condensation reaction catalyzed by DERA enzyme, which simplifies the synthesis process, but there are still many problems, such as the dosage of enzyme is too large, and each gram of isolated product needs 200mg DERA enzyme, which makes the cost of the method too high, and increases the difficulty of product separation; at the same time, due to the high concentration of substrate, DERA enzyme is easily inactivated, so that the substrate concentration of the catalytic reaction is limited
Greenberg [5] et al partially solved the problem of substrate inhibition through the fed-batch fermentation process, but did not fundamentally solve the problem of DERA enzyme inactivation caused by high substrate concentration

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1 Construction of E.coli mutant deoC library

[0039] Using a random mutagenesis kit, multiple reactions were performed by changing the concentration of MnSO4, thereby introducing 1 to 3 point mutations into the E.coli deoC gene (SEQ ID No.3), making 1 to 2 amino acids in the amino acid sequence of the DERA enzyme is replaced. To amplify the E.coli deoC gene (SEQ ID No.3) encoding the E.coli DERA enzyme (SEQ ID No.4), use primers DAI13600 (SEQ ID No.1) and DAI13465 (SEQ ID No.2) respectively as forward and reverse primers. Both primers contained sites compatible with the PCR-amplified deoC gene fragment obtained by site-directed recombination cloning using Gateway Technology. The gel electrophoresis patterns of the PCR amplification products are as follows: figure 1 shown.

[0040] Sequence SEQ ID No.1 of the forward primer (DAI13600):

[0041] 5’‐GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CGA AGG AGA TAG AAC CAT GAC TGA TCT GAA AGC AAG CAG CC‐3’

[0042] The...

Embodiment 2

[0045] Example 2 Expression of DERA enzyme

[0046] Expression of the mutated deoC gene in a deep-well microtiter plate: According to the mutant bacteria obtained in Example 1, select mutant colonies and inoculate them into 200 μl 2×YT medium (containing 100 μg / ml ampicillin) in a microtiter plate (MTP) , the culture condition is 37°C, and the time is 1 day. Then 100 μl of the above preculture was inoculated into a deep-well plate containing 500 μl of expression culture (2×YT, 100 μg / ml ampicillin, 1 mM IPTG), and then cultured on a shaker at 25°C for 24 hours. The SDS-PAGE electrophoresis pattern of the expression product is as follows: figure 2 shown. The expression of the unmutated deoC gene (SEQ ID No.4) under the same conditions was used as a control group.

Embodiment 3

[0047] Example 3 Preparation of Fluorescent Substrate

[0048] The substrate is a conventional methyl-5-tosyl-2-deoxynucleotide. Dissolve 12.75 g of tosylate in 75 ml of DMF, then add 11.86 g of K to the resulting solution 2 CO 3 and 9.29 g of 4-methyl-7-hydroxycoumarin (4-methylumbelliferone, 4-MU). After the above mixture was stirred at 75°C for 16 h, 300 ml of water was added and extracted twice with 200 ml of ethyl acetate. The organic phase was back-extracted with 100ml of 0.1M NaOH, and the organic phase was dried with sodium sulfate. The crude extract obtained by concentrating the organic phase was dissolved in a mixture of 25ml of acetonitrile and 100ml of water, then 2.5g of ion exchange resin Dowex50WX8-100 was added, stirred at room temperature for 1.5h, and then methanol was distilled off under reduced pressure. After 2 days, the mixture was filtered, concentrated under reduced pressure, and purified with a silica gel column, using gradient elution from 100% et...

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Abstract

The invention provides a 2-deoxy-D-ribose-5-phosphate aldolase (DERA enzyme) which is capable of catalyzing an aldol condensation reaction at high performance. Compared with a wild type escherichia coli DERA enzyme, the 2-deoxy-D-ribose-5-phosphate aldolase has improved substrate resistant ability and more efficient catalytic ability. If the DERA enzyme provided by the invention is used as a catalyst, the problem of the inactivation of the DERA enzyme caused by a high-concentration chloroacetaldehyde substrate can be solved, and simultaneously, the volume of addition of the enzyme in the production process is reduced. The invention also provides a nucleic acid for coding the DERA enzyme, a recombinant expression vector containing the nucleic acid, a recombinant expression transformant containing the recombinant expression vector, a method for preparing a recombinant DERA enzyme and a catalyst for forming an optically active statin intermediate through catalysis.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to an aldolase mutant and its gene, a recombinant expression vector and a recombinant expression transformant containing the gene, its recombinant enzyme and a preparation method of the recombinant enzyme, and the enzyme as a catalyst Application in the preparation of optically active statin intermediates by asymmetric aldol condensation. Background technique [0002] statins [1] It is a new type of cholesterol-lowering drug developed in the 1980s. It acts as a 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reduction inhibitor. By binding to the enzyme competitively, HMG- CoA cannot be transformed into mevalonate, an endogenous cholesterol raw material, thereby blocking the synthesis of cholesterol and reducing cholesterol levels. In addition, statins also have important functions such as lowering triglycerides and increasing high-density lipoprotein levels, so...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P1/00C12R1/19
Inventor 罗煜丁时诚瞿旭东
Owner ABIOCHEM BIOTECH CO LTD
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